The overall goal of this project is to identify molecular mechanisms that control the guidance of the oculomotor nerve. The oculomotor nerve innervates four of the six eye muscles. How the nerve wires up to each muscle is fundamental to the precise timing and coordination of motor signals, and drive the sophisticated set of eye movements required for vision. The oculomotor system is highly susceptible to developmental errors, causing eye movements or alignment in 2-5% of human infants. However, the basic mechanisms of how the oculomotor nerve navigates to the eye and targets specific muscles remain largely unknown. Our preliminary studies have identified the Slit/Robo repulsive guidance cues system as key regulators of several steps in oculomotor development, including axon projections to the eye, axon interactions with muscle precursors, and a unique motor neuron cell body migration across the midline of the midbrain. In the proposed project, we will use a range of complementary approaches, including studies of oculomotor nerve development in mutant mice, axon responses in culture, and transcriptomic analysis to identify new markers and candidate regulators of guidance. The aims of the project will be: 1) Determine the role of Slit/Robo signals in guiding the oculomotor nerve to the eye muscles; 2) Determine how another guidance system, the Semaphorin/Neuropilin signals, guide the oculomotor nerve, and how these signals interact with Slit/Robo signals. 3) Determine how Slit/Robo repulsion controls the midline migration of a specific subset of oculomotor neurons. Overall, this study will define a new experimental model for understanding the molecular basis for neuromuscular targeting, and identify novel mechanisms for the development of the oculomotor system